Antibiotic PA-39504-X1 and production thereof

ABSTRACT

A new antibiotic having β-lactamase inhibitory activity, PA-39504-X 1  of the formula: ##STR1## being useful as a medicament and a veterinary drug for inhibiting the growth of gram-positive and gram-negative pathogenic microorganisms and a process for preparing the same, being characterized by cultivating Streptomyces argenteolus PA-39504 or Streptomyces tokunonensis PA-31088 in a suitable medium and recovering PA-39504-X 1  from the cultured broth.

This invention relates to a new antibiotic, PA-39504-X₁ and the processfor preparing the same by cultivating a strain of Streptomycesargenteolus in a suitable medium and recovering PA-39504-X₁ from thecultured broth.

Said new antibiotic PA-39504-X₁ inhibits the growth of bothgram-positive and gram-negative pathogenic microorganisms. Furthermore,it shows a wide range of β-lactamase inhibitory activity.

The antibiotic PA-39504-X₁ has the following physicochemical properties.

(a) Mass spectrum of the methyl ester: m/e: 341 [M+H-16].

(b) Circular dichroism spectrum (in water): λ(nm) [θ] 360 (0), 320(-560), 267 (-6410), 245 (0), 240 (+1490), 230 (0), 220 (-2300), 210(0).

(c) Ultraviolet absorption spectrum (in 0.1 M phosphate buffer solution(pH 7.0)): λ_(max) ^(H).sbsp.2^(O) 240, 318 nm.

(d) NMR spectrum (in D₂ O at 5° C.): δ_(ppm) ^(D).sbsp.2^(O) 2.29,3.4-3.6, 3.8-4.0, 4.57, 5.4.

From the above data and other experimental results, the antibioticPA-39504-X₁ is identified to be3-[(2-acetamidoethyl)sulfinyl]-6-(1-hydroxymethylethylidene)-7-oxo-1-azabicyclo[3,2,0]hept-2-ene-2-carboxylicacid, the chemical structure of which is as follows: ##STR2##

Various epithienamycins and olivanic acids with β-lactamase inhibitoryactivity are known (The Journal of Antibiotics 32, 961-963 (1979)).However, PA-39504-X₁ is different from those antibiotics in thestructure, namely in the possession of 1-hydroxymethylethylidene at the6 position of 7-oxo-1-azabicyclo[3.2.0]hept-2-ene. Accordingly, it isconcluded that PA-39504-X₁ is a new antibiotic belonging to thienamycinantibiotics.

The antibiotic PA-39504-X₁ has the following biological properties:

(a) Anti-bacterial activity:

    ______________________________________                                                            Minimum                                                                       Inhibitory                                                                    Concentration                                             Test Bacteria       (μg/ml)                                                ______________________________________                                        Staphylococcus aureus 209P JC-1                                                                   6.25                                                      Streptococcus pneumoniae I                                                                        0.78                                                      Escherichia coli NIHJ JC-2                                                                        3.13                                                      Klebsiella pneumoniae SRL-1                                                                       6.25                                                      Klebsiella sp. 363 (R)                                                                            12.5                                                      Proteus mirabilis PR-4                                                                            6.25                                                      Enterobactor cloacae 233                                                                          12.5                                                      ______________________________________                                    

Note: inoculum size 10⁶

(b) β-Lactamase inhibitory activity

    ______________________________________                                                               Minimum                                                                       Inhibitory                                                                    Concentration                                          β-Lactamase Producing Bacteria                                                                  (μg/ml)                                             ______________________________________                                        1.     Cephalosporinase producing                                                    Enterobacter cloacae 92                                                                           0.5                                                2.     Penicillinase producing                                                       Klebsiella sp. 363  0.125                                              ______________________________________                                    

The antibiotic PA-39504-X₁ is produced by a microorganism belonging tothe genus Streptomyces. A microorganism was isolated from a soil sampleand tentatively named Streptomyces sp. strain No. PA-39504. Themicroorganism has been deposited in the Fermentation Research Instituteof Agency of Industrial Science & Technology, Yatabe-machi, IbaragiPref. Japan, 300-21 under the accession number FERM-P No. 5265 sinceNov. 5, 1979 and in American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md. 20852, U.S.A. under the accession number, ATCC No.31589 since Dec. 4, 1979.

Furthermore, PA-39504-X₁ can be produced by Streptomyces tokunonensisPA-31088 of which characteristics are disclosed in Japanese PatentPublication (Not-examined) No. 55-136282 published on Oct. 23, 1980. Thestrain has been deposited in Fermentation Research Institute under theaccession number FERM-P No. 4843 since Feb. 26, 1979 and in AmericanType Culture Collection under the accession number 31569 since Sept. 19,1979.

The microorganism Streptomyces sp. strain No. PA-39504 has the followingcharacteristics:

(a) Morphological Properties (cultured on Bennett's agar medium at 28°C. for 14 days)

The microorganism grows well on Bennett's agar medium and forms aerialhyphae abundantly. Aerial hyphae branch simply and the ends form loopsor short spirals. The aerial hyphae on the medium are brownish gray andthe substrate hyphae are pale yellowish brown. No soluble pigment isproduced. The surface of the spore is smooth under electron microscopyand the spores are short cylindrical. Any of sporangium, flagellatedspore or sclerotium is not observed. Split by fragmentation in substratehyphae is not observed.

(b) Physiological properties

    ______________________________________                                        Liquefaction of gelatin  Negative                                             Hydrolysis of starch     Positive                                             Tyrosinase reaction      Negative                                             Production of melanoid pigment                                                                         Negative                                             Peptonization of milk    Positive                                             Coagulation of milk      Negative                                             ______________________________________                                    

(c) Utilization of sugars

Good growth: L-arabinose, D-xylose, D-glucose D-fructose, inositol,L-rhamnose

No growth: sucrose, raffinose, D-mannitol

(d) Growth temperature

10° C.: fair growth but few aerial hyphae are formed.

28° C.: good growth with well formed aerial hyphae.

37° C.: no growth

45° C.: no growth

From the above properties, it is clear that the strain PA-39504 belongsto the Genus Streptomyces. The properties were elucidated with thedescription in "The Actinomycetes" 2 (1961) by Waksman, "InternationalJournal of Systematic Bacteriology" by Shirling and Gottlieb(International Streptomyces Project) 18 (1968), 19 (1969) and 22 (1972),"Bergey's Manual of Determinative Bacteriology" 8th edition (1974) andother published literature. It was concluded that the strain PA-39504belongs to Streptomyces argenteolus and is designated Streptomycesargenteolus PA-39504. The strains PA-39504 and PA-31088 have beendeposited in the Fermentation Research Institute and American TypeCulture Collection as noted above. This invention includes all naturaland artificial mutants and variants of both the above-describedmicroorganisms as long as it produces PA-39504-X₁ and cannot be clearlydistinguished from the above-described microorganisms. The artificialproduction of mutants may be accomplished by a conventional proceduresuch as X-ray or ultraviolet ray irradiation or treatment with nitrogenmustards, 4-nitroquinoline N-oxide, N-methyl-N'-nitro-N-nitrosoguanidineand other mutagens.

The production of PA-39504-X₁ is carried out by cultivating thePA-39504-X₁ producing strain in an enriched medium under aerobicconditions, whereupon PA-39504-X₁ is isolated from the cultured broth. Ageneral method of preparing PA-39504-X₁ is as follows:

The conditions of fermentation and the composition of the medium followthe usual known procedure for producing antibiotics. The composition ofthe medium may be varied over a very wide range. It essentially consistsof carbon sources, nitrogen sources and inorganic elements. Vitamins,precursors and other materials to stimulate the fermentation may beadded, if necessary. Examples of suitable carbon sources are glucose,starch, dextrin, glycerol, molasses, organic acids and the like. Theymay be used singly or in combination. Examples of nitrogen sources aresoybean meal, corn steep liquor, meat extract, yeast extract, cottonseed flour, peptone, wheat germ, ammonium sulfate and ammonium nitrate,which may be used singly or in combination. The inorganic elements maybe selected from, for example, calcium carbonate, sodium chloride,potassium chloride, magnesium sulfate, cobalt chloride, variousphosphates and the like. They are added to the medium if the occasiondemands. Liquid media are preferred for production on a large scale.

Fermentation may be carried out under aerobic or submerged aerobicconditions. A submerged aerobic culture is preferable. The pH of themedium may be adjusted to about 5.5 to 8.5. A buffering agent such ascalcium carbonate may be added to the medium if the pH of the mediumvaries during the fermentation.

The temperature may be kept at about 20° to 40° C., more preferably atabout 25° to 32° C., during fermentation. The fermentation perioddepends on the scale. It takes about 20 to 80 hours on a large scale. Ifexcessive foaming takes place during the fermentation, antifoamingagents such as vegetable oil, lard oil, and polypropylene glycol may beadded to the medium prior to or in the course of fermentation.

The antibiotic PA-39504-X₁ can be isolated from fermentation broth in aper se conventional manner. There may be employed any conventionalmethod such as filtration, centrifugation, adsorption and desorptionwith ion-exchange resins, chromatography with various active adsorbents,extraction with suitable solvents and the like. The procedures may becombined in appropriate order. During the isolation procedure,PA-39504-X₁ may be converted into the salt with a suitable base and asuitable stabilizing agent may be added in order to avoid thedecomposition.

The antibiotic PA-39504-X₁ can be converted into the pharmaceuticallyacceptable salts for use as medicament and veterinary drug. There areexemplified sodium, potassium, calcium, barium salts and the like.

The antibiotic PA-39504-X₁ is useful as a medicament, a veterinary drug,or as a sterilizer being effective against gram-positive andgram-negative bacteria including β-lactamase-producing strains.Therefore, PA-39504-X₁ and its pharmaceutically acceptable salts may beorally or parenterally administered to humans or animals. The antibioticmay be made into tablets, capsules, powder or the like in admixture withdiluents, stabilizing agents, preservatives, detergents and the like fororal administration. Further, it may be administered in forms of aninjection preparation, ointment or suppositories.

The dosage of PA-39504-X₁ is generally about 1/10th to several timesthat of cefalotin, although this is variable depending on the purpose ofthe treatment. For example, the daily dosage for a human adult is about0.1 to about 30 g for a subcutaneous injection.

PA-39504-X₁ has a strong β-lactamase inhibitory activity andsynergetically increases the anti-bacterial activity of known β-lactamtype antibiotics against β-lactamase producing bacteria. Therefore,PA-39504-X₁ may be used in combination with the well-known antibioticsof the β-lactam type such as penicillins, e.g. benzylpenicillin,phenoxymethylpenicillin, carbenicillin, ampicillin, amoxicillin and thelike, and cephalosporins, e.g. cefaloridine, cefalotin, cefazolin,cefalexin, cefacetrile, cefamandole, cefradine, cefaloglycin, cefoxitin,cefapirin and the like.

The following examples are given solely for the purpose of furtherexplanation and is not to be construed as limiting of the presentinvention, many variations of which are possible.

EXAMPLE 1 (a) Fermentation process

Medium S-1: soluble starch 0.5%, glucose, 0.5%, polypeptone 0.5%, meatextract 0.5%, yeast extract 0.25%, sodium chloride 0.25%, and deionizedwater (pH 7.0 before sterilization).

A slant of seed culture of Streptomyces sp. PA-39504-X₁ (Ferm-P No.5265) is inoculated into a 2-liter Erlenmeyer flask containing 800 ml ofMedium S-1 of the above composition and incubated at 28° C. for 48 hoursunder stirring at 180 r.p.m.

The germinated seed broth (800 ml) is inoculated into a 30-liter jarcontaining 20 liters of Medium S-1 and incubated for 64 hours at 28° C.with aeration of 20 liters per minute and under an internal pressure of0.2-0.3 kg/cm² and stirring at 200 to 300 r.p.m.

(b) Isolation process

The fermentation broth of the above process is centrifuged by aSharpless centrifugal separator. The supernatant fluid (122 liters) iscooled to 10° C., adjusted to pH 7.0 and passed through a column of 10liters of Dowex 1×2 (Cl⁻) (by Dow Chemical Co., USA) at a rate of 500 mlper minute. The column is eluted with a 5% sodium chloride-cooleddeionized water solution. The fractions (18 liters) showinganti-bacterial activity as checked by the pulp disk diffusion methodwith Escherichia coli are collected, adjusted to pH 7.0, and passedthrough a column of 9 liters of Diaion HP-20 (by Mitsubishi Kasei Co.,)at a rate of 150 ml per minute. The column is eluted with cooleddeionized water containing 2% methanol. The active fractions (9.6liters) are collected, adjusted to pH 7.0 and lyophilized to give acrude powder (14.25 g).

(c) Purification Process

The crude powder of PA-39504-X₁ (13.8 g) prepared above is dissolved inwater (30 ml) and applied to a column of about 500 ml of Dowex AG 1×2(Cl⁻). The column is eluted with 0.005 M ammonium chloride (adjusted topH 7.0 with ammonium hydroxide) and 0.4-3.0% sodium chloride solutionsby gradient method. The eluates are separated into five fractions bychecking the activity by pulp disk diffusion method with Escherichiacoli. Each fraction is desalted with a column of Diaion HP-20, condensedand lyophilized. The crude powder (776 mg) obtained from the secondfraction is dissolved in water (3 ml) and applied to a column of 120 mlof Dowex AG 1×2 (Cl⁻). The column is eluted with 0.05-0.5 M phosphatebuffer solution (pH 7.0) by gradient method. The active fraction isdesalted in the same manner as noted above and lyophilized to give apowder (172 mg). The powder is dissolved in water (0.5 ml) and appliedto a column of LiChroprep RP-18 (by Merck E. AG) (20 mm×50 cm). Thecolumn is eluted with 0.05 M phosphate buffer solution (pH 7.0). Theactive fractions are desalted and lyophilized to give a powder (60 mg).

The resultant powder (60 mg) is dissolved in 0.05 M phosphate buffersolution (pH 7.0) and applied to high performance liquid chromatographywith a column (10 mm×30 cm) of Nucleosil-5-C₁₈ (M. Nargel Co., WestGermany) with the above buffer solution as eluent. The active fractiondesalted and lyophilized to give a powder (16 mg).

The powder (16 mg) is applied to high performance liquid chromatographyon Nucleosil-5-C₈ (10 mm×30 cm) with 0.1 M phosphate buffer solution (pH7.0), desalting and lyophilization to give an amorphous powder (1.5 mg)of PA-39504-X₁.

EXAMPLE 2

PA-39504-X₁ is obtained by using Streptomyces tokunonensis PA-31088(FERM-P No. 4843) in the same manner as in Example 1.

What is claimed is:
 1. An antibiotic PA-39504-X₁ of the formula:##STR3## or a pharmaceutically acceptable salt thereof.
 2. Theantibiotic as claimed in claim 1, of which the physicochemicalproperties are as follows:(a) Mass spectrum of the methyl ester: m/e:341 [M+H-16]. (b) Circular dichroism spectrum (in water): λ(nm) [θ] 360(0), 320(-560), 267 (-6410), 245 (0), 240 (+1490), 230 (0), 220(-2300),210 (0). (c) Ultraviolet absorption spectrum (in 0.1 M phosphate buffersolution (pH 7.0)): λ_(max) ^(H).sbsp.2^(O) 240, 318 nm. (d) NMRspectrum (in D₂ O at 5° C.): δ_(ppm) ^(D).sbsp.2^(O) 2.29, 3.4-3.6,3.8-4.0, 4.57, 5.4.